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Reproductive Technology |
Research Institute for Animal Breeding and Nutrition,3 Department of Cell Biology, Herceghalom, Hungary
Biometris,4 Wageningen, The Netherlands
Alta Genetics, Inc.,5 Kleine Huisjes, The Netherlands
Institute of Biomembranes6 and Graduate School Animal Health,7 Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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acrosomal membranes, cryopreservation, egg yolk, plasma membrane
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One of the most used fluorochrome combinations for simultaneous evaluation of plasma membrane integrity (i.e., viability) and acrosome integrity are fluorescein isothiocyanate-conjugated pea (Pisum sativum) agglutinin (FITC-PSA) and propidium iodide (PI) [2]. On nonpermeabilized spermatozoa, FITC-PSA provides information regarding the integrity of the acrosome: Sperm cells with an intact acrosome will have no fluorescence, and cells with a reacted or damaged acrosome will show green fluorescence. Propidium iodide is a DNA-specific stain that cannot enter the intact plasma membrane and, therefore, is used as a dead-marker counterstain. This double-staining for sperm viability and acrosome integrity is relatively reliable for fresh and in vitro-capacitated sperm, because sperm cell particles can easily be distinguished from nonsperm events by their specific forward- and sideways-scatter properties (e.g., in dogs [3]). For frozen-thawed sperm, the main problem is that many of the egg yolk particles have scatter properties similar to those of sperm cells that trouble the elimination of nonsperm events by scatter gating enormously [4]. Such egg yolk particles, like live acrosome-intact sperm cells, have low fluorescence and, therefore, will be assessed as live acrosome-intact sperm using the PI/FITC-PSA double-labeling method. Therefore, when using the PI/FITC-PSA double-staining protocol, complete removal of yolk particles from thawed sperm suspensions is required for accurate analyses of sperm integrity after cryopreservation. However, this separation is most likely coupled to induction of sperm deterioration. Obviously, inappropriate estimation of sperm survival after cryopreservation may affect mammalian and human artificial reproduction technologies (and the development of new freeze-thaw protocols).
To distinguish egg yolk particles from sperm cells without the necessity of removing egg yolk by washing, Garner et al. [5] reported a simple stain combination for evaluating viability: The two stains, SYBR-14 (a green, membrane-permeable stain) and PI (a red, membrane-impermeable counterstain) have the same cellular target, the sperm DNA. Because this stain combination has several advantages, such as quickness, simplicity, and excitation with visible light, our approach was to find a suitable acrosome stain that could be used in combination with the well-validated SYBR-14/PI staining. In contrast to live or deteriorated sperm cells, egg yolk particles do not contain DNA and, therefore, will not be stained by SYBR-14/PI and, in theory, can easily be omitted from flow cytometric analyses (i.e., gated out) [6].
In the present study, we extended this live-dead dual-staining (SYBR-14/PI) with a third stain: Phycoerythrin (PE)-conjugated PNA. This probe acts on sperm acrosomes in a manner identical to that of FITC-PSA [2] and, therefore, can be used to detect the integrity of the sperm acrosome. We decided to use PE-peanut (Arachis hypogea) agglutinin (PE-PNA) instead of the PE-PSA, because the egg yolk particles showed some affinity for PSA [7]. The PE fluorescent moiety was used, because its fluorescence emission can be measured independently from that of PI or SYBR-14.
The present study reports on the applicability of an SYBR-14/PE-PSA/PI triple-staining technique to simultaneously assess the plasma membrane integrity and the acrosome integrity of frozen-thawed bovine sperm cells in the presence of egg yolk particles using a flow cytometer. So far, most flow cytometric analyses of frozen-thawed sperm samples have been done after removal of egg yolk by centrifugation. Most likely, the washing of thawed sperm samples induces further deterioration as an artifact, which may be unrelated to the postinsemination behavior of frozen-thawed sperm in the cow (thawed sperm is not washed before insemination). Use of the triple-labeling technique for assessing the integrity of a thawed sperm specimen immediately after staining (i.e., without any intervening sperm-processing steps) in relation with sperm cryobiology and its predictive value for artificial reproductive technologies is discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LIVE/DEAD Sperm Viability Kit (L-7011; SYBR-14 and PI) was purchased from Molecular Probes, Inc. (Eugene, OR). Peanut agglutinin conjugated with phycoerythrin (Phycoprobe R-PE-PNA, P44) was purchased from Biomeda Corp. (Foster City, CA). Dimethyl sulfoxide (DMSO; D-5879) and peanut agglutinin conjugated with fluorescein isothiocyanate (FITC-PNA; L-7381) were purchased from Sigma Chemical Co. (St. Louis, MO). CellWash optimized PBS (catalog no. 349524) was purchased from Becton Dickinson (San Jose, CA).
Semen Samples
Bull semen was collected and diluted to a final concentration of 60 million cells/ml in a Tris-homogenized egg yolk extender made up of 0.2 M Tris-HCl, 0.08 M citric acid monohydrate, 0.07 M fructose, 6% (v/v) glycerol, and 20% (v/v) egg yolk at pH 6.8. After initial dilution of the bull semen at 35°C, the semen was slowly cooled to 5°C over a period of 90 min, subsequently packaged at 60 million sperm/ml in 0.25-ml French-straws, and frozen in nitrogen vapor following the IMV Digit-cool standard curve for bovine semen package in 0.25-ml French-straws (IMV, l'Aigle, France). For semen analysis, straws were thawed for 1 min at 37°C in a waterbath. For flow cytometric analysis, 100 µl of thawed semen samples were added to 900 µl of CellWash optimized PBS in Falcon tubes (Becton Dickinson).
Triple-Staining of Frozen-Thawed Sperm Samples Containing Egg Yolk
To investigate the repeatability of the SYBR-14/PE-PNA/PI assay, we used 10 bulls, two ejaculates per bull, three straws per ejaculate, and two measurements per straw.
At a final concentration of 100 nM SYBR-14 working solution (component A of the LIVE/DEAD Sperm Viability Kit, diluted 10-fold with DMSO), 2.5 µg/ml of PE-PNA solution (1 mg/ml of stock solution in a buffer composed of 3.0 M ammonium sulfate, 50 mM sodium phosphate, and 0.05% sodium azide, pH 7.0, and also containing 1 mM [Ca2+] and [Mn2+] ions) and 12 µM PI stock solution (undiluted component B of LIVE/DEAD Sperm Viability Kit) were added to 1 ml of diluted semen. Samples were mixed and incubated at 37°C for 10 min and then remixed just before analysis. Stained sperm suspensions subsequently were run through a flow cytometer (FACSCalibur; Becton Dickinson). The three dyes were excited in the flow cytometer using a 488-nm argon excitation laser. Nonviable cells become PI positive, and their red fluorescent signal is detected using fluorescence detector 3 (detects emitted photons of a wavelength >670 nm). Living cells are SYBR-14 positive, and its green fluorescent signal is detected using fluorescence detector 1 (detects emitted photons in the wavelength of 515–545 nm). Finally, acrosome-damaged cells are stained positive for PE-PNA, and its orange fluorescent signal is detected using fluorescence detector 2 (detects emitted photons in the wavelength range of 561–583 nm). The three dyes have minimal emission overlap. Adjustment of compensation values for the three emission detectors used was done according to the guidelines of Roederer (available at http://www.drmr.com/compensation). Nonsperm events were gated out of analyses as judged on scatter properties as detected in the forward-scatter and sideways-scatter detector, respectively (scatter-gated sperm analysis). On top of this, events with scatter characteristics similar to sperm cells but without reasonable DNA content (very weak SYBR-14 or PI staining) were also gated out (double-gated sperm analysis).
The cytometer was calibrated each day with CaliBRITE 3 three-color kit and CaliBRITE APC beads (Becton Dickinson) using the FACSComp 4.1 automatic calibration software. The cytometer was used at the "low" flow rate (12 µl/min). The recording of scatter and fluorescent properties of all events stopped when 10 000 double-gated events were recorded. Two dimensional plots of sideways- and forward-scatter properties as well as of PE-PNA fluorescence or SYBR-14 versus PI fluorescence were drawn. For the PE-PNA versus PI dot plots, subpopulations were divided by quadrants, and the frequency of each subpopulation was quantified. The staining patterns were verified by inspecting sperm samples under an epifluorescence microscope (Leica DM-LB, Leica GmbH, Heidelberg, Germany) equipped with a dual blue/green filter set (Leica 11513803).
Comparison with Conventional Dual-Staining with PI and FITC-PNA
To estimate the measurement error caused by the egg yolk contamination in the live, acrosome-intact sperm cell population when assessed by FITC-PNA and PI, we carried out paired measurements on 14 randomly selected frozen-thawed semen samples using SYBR-14/PE-PNA/PI and FITC-PNA/PI. Measurements were done immediately after thawing and after a 3-h incubation at 37°C (stress test).
At a final concentration of 1 µg/ml of FITC-PNA working solution [8], 1 mg/ml of stock solution with DMSO (working solution further diluted 5-fold with PBS) and 12 µM PI stock solution (undiluted component B of LIVE/DEAD Sperm Viability Kit) were added to 1 ml of diluted semen. Samples were mixed and incubated at 37°C for 5 min and remixed just before analysis.
Flow cytometric analyses were carried out as described above with the exception that FITC-PNA fluorescence was detected at 515–545 nm Fluorescence detector 1 (Fl 1). Nonsperm events were gated out of analyses as judged on scatter properties only. Fluorescent data of all events were collected until 10 000 gated events were recorded. Two-dimensional plots of FITC-PNA versus PI fluorescence events were drawn. Subpopulations were divided by quadrants, and the frequency of each subpopulation was quantified.
Statistical Analysis
Repeatability and method-agreement analyses [9] were done with Microsoft Excel 97 software (Microsoft, Redmond, WA). To assess the repeatability of a method, we calculated the differences between pairs of repeated measurements and the mean of these differences (d), which is an estimate of the bias of the first measurement relative to the second. As a measure of repeatability, the British Standards Institution repeatability coefficient [10] was calculated as twice the SD of the differences. It is used as an indication of the maximum difference likely to occur between repeated measurements.
Method-agreement analysis between the two staining protocols was done by the same statistical procedure. The mean of the differences between the paired measurements on the same samples (d) was calculated to estimate the average bias of one method relative to the other. The 95% limits of agreement were calculated as d ± 2SD, where SD is the standard deviation of the differences between paired measurements [10].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immediately after thawing, sperm cells were stained with PI (deteriorated cells fluoresce red at the DNA-containing nucleus; living cells are not labeled) and FITC-PNA (acrosome-intact cells are not labeled; acrosome-damaged/reacting cells fluoresce green at the outer acrosomal membrane that is exposed). After 10 min, the labeled cells were assessed by flow cytometry in terms of viability and acrosome integrity (Fig. 1). A considerable amount of nonsperm particles ran through the flow cytometer. Using two-dimensional sideways- and forward-scatter dot plots (Fig. 1A), it was possible to gate out nonsperm particles with scatter properties differing from those of sperm cells (Fig. 1B). Nongated compared to gated analysis of the data obtained severely affects the measured percentage of surviving acrosome-intact sperm (Fig. 1, C and D, respectively). Sperm cells incubated for 3 h in dilution buffer were also analyzed with and without use of the scatter gate (Fig. 1, F and E, respectively). The majority of egg yolk particles were removed from analysis by this scatter gate; however, especially in Figure 1F, a subpopulation of nonsperm events (most likely larger egg yolk particles/aggregates) obviously remained, interfering with this conventional flow cytometric assay. The average difference between repeated measurements of the dual-staining (d) was -0.49% (SD = 1.82%), with the British Standards Institution repeatability coefficient (2SD) being 3.64%, indicating a good repeatability (Fig. 2).
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SYBR-14/PE-PNA/PI Triple-Staining of Frozen-Thawed Bull Sperm in the Presence of Yolk Particles
Immediately after thawing, sperm cells were stained with PI (deteriorated cells fluoresce red at the DNA containing nucleus; living cells are not labeled), SYBR-14 (a membrane-permeable probe that fluoresces green on nuclei of living sperm cells but leaks out of deteriorated cells), and PE-PNA (acrosome-intact cells are not labeled; acrosome-damaged/reacting cells fluoresce orange at the outer acrosomal membrane that is exposed). After 10 min, the labeled cells were assessed by flow cytometry in terms of viability and acrosome integrity (Fig. 3). The fluorescent emission signals of the probes were selectively detected in their corresponding fluorescence detectors (photomultiplier tubes). Again, as was also depicted for the conventional dual-stain (Fig. 1), a majority of nonsperm events (yolk particles) could be removed from analysis by scatter gating (red events indicated in Fig. 3, A, D, and G). By creating two-dimensional dot plots of PI versus SYBR-14 fluorescence (Fig. 3, A–C), we noted three subpopulations of events analyzed by the flow cytometer. The first population was nonlabeled, which indicates that these events did not contain DNA. The second population was SYBR-14 positive but PI negative, indicating that these cells were plasma membrane intact and contained DNA. The third population contained cells with PI fluorescence and no sign of SYBR-14 fluorescence, indicating that these cells contained DNA but were deteriorated after freeze-thawing. By gating out the nonfluorescent (i.e., no-DNA-containing) particles, we were able to get very clear two-dimensional dot plots of PI versus PE-PNA (green cells in Fig. 3, D–I). The remaining black particles indicate non-DNA-containing particles that probably represent aggregated egg yolk particles because of the larger scatter signals. Considerable amounts of nonsperm particles ran through the flow cytometer. The middle panels of Figure 3 can be compared with the results obtained by dual-staining (Fig. 1). However, as depicted in Figure 3, B, E, and H, a substantial subpopulation of nonsperm events interfere in the scatter-gated data. The results in Fig. 3, C, F, and I show that the percentage of surviving acrosome-intact sperm recorded in SYBR-14/PI gating is appreciably lower than that for recorded for scatter-gated data (compare Fig. 3, B, E, and H vs. C, F, and I). Sperm cells incubated for 3 h in dilution buffer were also analyzed with and without use of the scatter gate or SYBR-14/PI (Fig. 3, G–I). The majority of egg yolk particles were removed from analysis by this scatter gate; however, a subpopulation of nonsperm events (most likely larger egg yolk particles/aggregates) obviously remained, interfering with this conventional flow cytometric assay (Fig. 3, H and, to a lesser extent, E). All nonsperm events were eliminated from analyses by SYBR-14/PI gating. This triple-labeling protocol therefore enables direct staining and flow cytometric analyses of frozen-thawed sperm cells without requiring any wash steps to remove unbound probe (required for microscopy) or egg yolk particles (easily gated out). The average difference between repeated measurements of the triple-staining (d) was -0.7% (SD = 1.3%), with the British Standards Institution repeatability coefficient (2SD) being 2.6%, indicating a high repeatability (Fig. 4).
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Statistical Comparison of the Dual- and Triple-Staining Data
Average numbers (mean ± SD) of acrosome-intact and living sperm cells in the 14 straws tested are indicated in Table 1. As method-agreement analysis showed, measurements by FITC-PNA/PI were, on average, 6.3% larger than measurements by triple-staining (SD = 4.65%) (Fig. 5A). The difference between the means of the dual- and triple-staining measurements became larger (15%) after a 3-h incubation at 37°C. With average differences of d0 h = 4.21%, d3 h = 8.44% between repeated measurements of dual- and triple-staining measurements of 0 and 3 h, respectively (Fig. 5, B and C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In dual-staining, sperm cells were first labeled with FITC-PNA and PI, and sperm-specific scatter events were gated from nonsperm scatter events. In the triple-stain method, we noted a substantial subpopulation of sperm-"specific" scatter events that were not labeled for either SYBR-14 or PI and, therefore, that could not be considered as sperm cells. These nonsperm events cannot be gated out using the dual-staining protocol. Even using stringently homogenized egg yolk (to create more homogeneously sized egg yolk particles) resulted in more fuzzy two-dimensional PI and FITC-PNA dot populations. The fuzzy interference of nonsperm events with sperm-like scatter properties turned out to be the cause of this fuzzy interference. The dual-staining procedure resulted in a consistent underestimation of the percentage of acrosome and plasma membrane deterioration immediately after freezing or after 3-h incubation in diluted sperm after removal from the straws when compared to the triple-staining procedure. Measurements obtained by the dual-staining procedure are, on average, 6.3% larger than those obtained by the triple-staining procedure. The difference between the paired measurements became larger after a 3-h incubation at 37°C (4.2% and 8.4% at 0 and 3 h, respectively.). This could be attributed to the higher proportion of events with sperm-specific scatter properties that appeared as slightly FITC-PNA positive in 3-h incubated sperm versus 0-h incubated sperm (indicated with arrows in Fig. 1, D and F, respectively). This fuzzy, diagonal subpopulation was not seen in double-gated, triple-stained sperm samples irrespective of postthawing incubation (see Fig. 3). The subpopulations indicated with arrows in Figure 1, D and F, may well represent small vesicles originating from the sperm plasma membrane and outer acrosomal membrane that vesiculize during the acrosome reaction (which explains the presence of FITC-PNA-binding sites [8]). If so, then such vesicles are not detected by the triple-staining method, because they do not contain DNA (i.e., are gated out for analyses because they are double-negative for SYBR-14 and PI).
Statistical analysis showed the SYBR-14/PE-PNA/PI triple-staining results to be highly repeatable: The British Standards Institution repeatability coefficient was 2.6%. The repeatability coefficient of the FITC-PNA/PI staining under similar experimental conditions was 3.6% (Fig. 2).
One question that must be addressed is whether the dead cells with PNA binding exhibited at an earlier stage an acrosome reaction or whether the PNA binding was a result of the loss of viability of these cells (the so-called false acrosome reaction [12]). Most likely, cryopreservation leads to acrosome damage in a subpopulation of sperm cells, and such cells also have disrupted plasma membranes. The small amount of acrosome-reacted cells that survived freeze-thawing was only 1.7% (detected at 0 h postthawing), and this number dropped to less than 0.5% after 3-h incubations at 37°C. Our data indicate that thawing does not specifically induce acrosome reactions in live bull sperm. It is generally accepted that when combining a viability and an acrosome stain, dead cells with a reacted acrosome show a "false" acrosome reaction. However, as stated for the fix-vital staining method [12] and for eosin/aniline blue staining [13, 14], it is difficult to rule out the other possibility that acrosome-reacted cells rapidly showed further deterioration. Therefore, the population of acrosome-reacted, dead sperm cells most likely is a mixture consisting of cells that died after the acrosome reaction (and, therefore, had a "true" acrosome reaction) and of cells that lost the acrosome at cell death (a "false" acrosome reaction).
Previous attempts have been made to discriminate sperm cells from egg yolk particles using the DNA marker probe SYTO-17 in combination with FITC-PNA and PI to shift the intact sperm population from the extender particles [7]. Furthermore, Pena et al. [4] developed a new fluorescent staining method using carboxy-SNARF-1, an intracellular pH indicator that stains the live cells orange, and those authors used this in combination with FITC-PNA and PI. However, the staining mechanism of SYTO-17 cannot be validated by the naked eye, which does not sense far-red emission photons. Related to this, the flow cytometer that is used must be equipped with a second excitation laser (excitation at 621 nm) for the optimal excitation. Carboxy-SNARF-1 has a different cellular target compared to PI and is less valuable for discrimination between nonsperm and sperm particles. The triple-staining method presented here only requires a simple flow cytometer equipped with one 488-nm argon laser and makes use of well-established DNA and acrosome dyes.
We may note that considerable differences exist between egg yolk preparations. In a separate experiment, we could even identify the firm stud where bull sperm had been cryopreserved in straws based on the scatter properties (unpublished results). Such variations will create variations in sperm-quality parameters as detected with the dual-staining protocol. Scatter-independent gating of nonsperm particles from sperm cells using combined SYBR-14/PI gating is, therefore, the method of choice to detect frozen-thawed sperm.
Besides damage at the level of the plasma membrane or acrosome, cryopreservation of sperm may induce damage to the mitochondria, which can be detected with mitotracker probes that sense the potential of the inner mitochondrial membrane. Depolarization of this membrane will distort the electron transport chain, the proton gradient, and thus aerobic ATP production in the midpiece of the sperm cell, rendering its motility characteristics inadequate. Different mitotracker probes have already been used to detect mitochondrial disruption in mammalian sperm [11, 15–17]. Therefore, we suggest that our triple-staining method should be extended to a quadruple-staining method in which mitochondrial functionality can be simultaneously assessed with the acrosomal and plasma membrane damage in the presence of egg yolk. The radiometric JC-1 dye [15, 17] is not suitable for such multicoloring experiments because of the broad-emission spectral properties of this mitochondrial-potential probe. However, the commercially available Mitotracker 633 deep red (Molecular Probes Europe BV, Leiden, Netherlands) can be used in combination with our triple-staining method. The flow cytometer requires a second excitation He/Ne laser with a 633-nm line, and the functional status of the mitochondria can be detected independently from the triple-staining assay described here at wavelengths greater than 680 nm.
In conclusion, this new triple-stain combination is highly repeatable and easy to use for routine artificial insemination or other assisted reproductive technologies, and it gives a more accurate estimation of the proportion of intact sperm cells. Frozen-thawed sperm specimens can now be directly stained and analyzed with a relatively simple flow cytometer. Classical removal of egg yolk, with the high risk that processing will cause sperm degeneration, is avoided by a triple-stain technique that allows immediate and noninvasive elimination of all nonsperm particles from flow cytometric analyses. This triple-staining assay will have major impact on cryobiological research of mammalian sperm.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: B.M. Gadella, Institute of Biomembranes, Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584 CM Utrecht, The Netherlands. FAX: 31 30 2535492; b.gadella{at}vet.uu.nl
Received: 24 September 2002.
First decision: 27 October 2002.
Accepted: 9 December 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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